double bond, eliminating the leaving group to generate a new resonant double bond. A second Michael

addition is then possible with another nucleophile on the same or different protein, resulting in a crosslinkage. Alternatively, during the second Michael reaction process, the first added nucleophile may

be eliminated reforming a double bond. A nucleophile may then undergo similar Michael addition

and the process continues. The reagent can be transferred from the initial site of protein attachment to

other groups until the most thermodynamically stable bond is formed. With the protein side chains of

In addition to the above categories of heterobifunctional cross-linkers, there are other heterobifunctional reagents that cross-link various nucleophiles. The Cyssor reagent, 2-methyl-N1-benzenesulfonylN4-bromoacetylquinonediimide (Appendix D.F.CXLIX), has been found to cross-link antibodies at

pH 8.0.72 This molecule is essentially an alkylating agent. Nucleophiles attack both the quinone ring

and the bromoacetyl group as shown in Figure 6.4. Although the nucleophiles in this particular reaction have not been identified, it is speculated that carboxyl groups of aspartate and glutamate and the

indolyl ring of tryptophan may serve as nucleophiles in addition to sulfhydryls and amino groups.

Compounds that contain an aldehyde group such as N-hydroxysuccinimidyl-p-formylbenzoate (HFB)

and methyl-4-(6-formyl-3-azidophenoxy)butyrimidate (FAPOB) (Appendix D.F.CL and CLI) will form

containing an phenylazido group, is also photosensitive and may be used as a photochemical agent. In

acrolein (Appendix D.F.CLIII), the aldehyde group may form a Schiff base with an amino group. Its

double bond is subject to Michael addition with different nucleophiles and has been shown to cross-link

collagen.77 Ishii et al.78 used model peptides and mass spectrometric techniques to show that crosslinking took place between amino groups and the side chain of histidine in the peptide. Compounds that

contain either trimethoxysilyl or triethoxysilyl moiety such as 3-glycidyloxypropyltrimethoxy silane and

CXXXIV and CXXXVI in Appendix D) are capable of reacting with hydroxyl groups of silica on glass

surfaces. The other end of the molecules containing reactive epoxide or isothiocyanates can react with

various nucleophiles, thus immobilizing the molecules as shown in Figure 6.5. These compounds have

been used for the preparation of bioconjugates and immobilization of biomolecules such as oligonucleotides, peptides, and proteins on the glass surface.79,80

Affinity labeling is a specially designed agent that will bind specifically to a desired location of a biomolecule, usually the active site. An example of an affinity bifunctional cross-linker is

EtO

CH2-N=C=S

EtO Si (CH2)3 NH-CO

EtO

Glass

Molecule-Nu:

OH

EtO

Glass

O

S

EtO

Si (CH2)3 NH-CO

CH2-N=C=S EtO

Si

(CH2)3 NH-CO

CH2-NH-C Nu-Molecule

EtO

EtO

Molecule-Nu:

O

OH

S

EtO

Glass

Glass

Si (CH2)3 NH-CO

CH2-NH-C

Nu-Molecule

EtO

FIGURE 6.5 Reaction mechanism of immobilization of macromolecules on to glass surface using N-(3triethoxysilylpropyl)-4-(isothiocyanatomethyl)cyclohexane-1-carboxamide. (Adapted from Misra, A. et al.,

Bioorg. Med. Chem. Lett., 18, 5217, 2008.)

202

Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

1-(4-methoxyphenyl)-3-acetamido-4-methoxyazetidin-2-one (Appendix D.F.CLVI), which is an analog of

β-lactam.81 It inhibits the class A β-lactamase from Bacillus cereus 569/H as an active site-directed inhibitor and cross-links ser 70 and lys 234 of the enzyme. However, the mechanism of reaction is unknown.

6.3 P

ROTEIN-PHOTOSENSITIVE HETEROBIFUNCTIONAL

CROSS-LINKING REAGENTS

Photosensitive heterobifunctional cross-linkers represent by for the largest portion of the heterobifunctional reagents. For a comprehensive list of these compounds, please see Appendix E. Because these

functionalities are inert until they are photolyzed, these reagents are first linked to the protein in the dark

through a group-directed agent as shown in Table 6.2. The labeled protein is then irradiated to activate

the photosensitive group, which reacts indiscriminatively with its environment as discussed in Chapter 4.

The photosensitive cross-linkers are generally classified according to the active species they produce,

for example, the nitrenes and carbenes.82 These reactive species are generated from various groups. As

discussed in Section 4.5, the most widely used photoreactive groups are azides, diazo moiety, benzophenone, diazonium salts, and diazirines. Only a few carbene-generating diazo reagents are used

for cross-linking,65,67,68,82–87 probably because of the ability of carbenes to undergo a variety of reactions including the very efficient reaction with water.82 In addition, the parent diazoacetyl compounds

are generally unstable, particularly at low pH and are reactive toward nucleophiles including carboxyl

groups.64 However, 3-phenyl-3-(trifluoromethyl) diazirine (TPD) is gaining popularity and has been

incorporated into photoaffinity labels and cross-linkers.88,89 The reasons are the unexpected stability of

the TPD three-membered ring and its ability to be photoreactivated with light over 350 nm to generate

carbenes, which can rapidly form cross-links to biomolecules with short photoirradiation times.

imines. Last, alkylazides are reactive and may undergo nucleophilic displacement reactions. For the

same reasons, acylazides are generally used as acylating agents rather than photoaffinity labels.90 Only

arylazides have been extensively used in photoactivatable cross-linkers.90–138 Aryl azides have a low

activation energy and can be photolyzed in the long UV region.21,82,139 The presence of electron-withdrawing substituents such as nitro and hydroxyl groups further increases the wavelength of absorption

into the 300 nm region.64 Arylnitrenes have a half-life on the order of 10 −2–10 −4 s,140,141 and, therefore,

the cross-linking reaction is expected to be terminated within a short time. Arylazides are susceptible

to reduction to amino groups. They are not stable in the presence of thiols. The half-life of arylazides

The benzophenone derivatives (LXII through LXV, CXI, CXII, and CXL through CXLIV in

Appendix E) constitute yet another class of photoaffinity labels.143–145 These compounds, as shown in

Chapter 4, can form covalent adducts on irradiation with nearby amino acid residues leading to crosslinking. Unlike the azides, which are irreversibly photolyzed in most cases, the excited triplet state of

benzophenones may be resistant to reaction with water and may revert back to the starting material if no

photoreaction takes place. Since benzophenones can be reexcited, their cross-linking efficiency can, in

principle, reach 100%.143,144 These compounds have been used to study virus-induced proteins,146 ribosomal proteins,147 troponin and tropomyosin,148 actin,149 thin filament proteins,150 and chymotrypsin.151

Another class of photosensitive reagents is nitrophenyl ether (Appendix E.B.CXIV and CXV).152

These compounds react quantitatively with amines at slightly alkaline conditions (pH 8) on irradiation with 366 nm light. The reaction involves the transfer of nitrophenyl group from the alcohol to the

amine as shown in Figure 6.6 for the cross-linking reaction of N-(maleimidomethyl)-2-(O-methoxyp-nitrophenoxy)carboamidopropane. With this compound, the 2-methoxy-4-nitrophenyl ether is